High density lipoprotein (HDL) plays a major role in cholesterol homeostasis and atherosclerosis. Yet our understanding of its structure, interactions with HDL-associated proteins, and alterations in function during atherosclerosis, remain poorly understood. We propose to use a multidisciplinary approach to better understand how distinct structural elements of HDL facilitate specific biological functions in reverse cholesterol transport (ROT) and may become dysfunctional through site-specific oxidative modification within human atherosclerotic plaque. We have developed novel tools for structurally, functionally and clinically characterizing dysfunctional forms of HDL and their involvement in human disease. We will employ these to achieve our overall major goals of: (i) defining HDL function and site-specific oxidative modifications within human atherosclerotic plaque that adversely impact upon normal lipoprotein function; and (ii) exploring the clinical utility of quantifying specific dysfunctional HDL forms in human clinical studies. We will achieve this with the following specific aims: Aim 1) To define important structural elements critical for nascent HDL particle interaction with LOAT and maturation into a cholesterol-ester laden spheroidal form, and to test the hypothesis that site-specific oxidative modifications of apoAl Tyr166, a known functional residue in the LOAT interaction site modified within human atherosclerotic plaque, generates both a dysfunctional form of HDL and identifies individuals at increased risk for atherosclerotic heart disease. Aim 2) To discover the structural modification induced by MPO-catalyzed oxidation on apoA1 of HDL that converts the particle into a pro-inflammatory form and to test the hypothesis that MPO-specific oxidative modification of HDL identifies individuals at increased risk for atherosclerotic heart disease. Collectively, the proposed studies represent an innovative and multidisciplinary approach designed to elaborate key structural elements of HDL that support specific atheroprotective functions and how site specific oxidative modifications to specific residues in vivo adversely impacts upon normal lipoprotein function and generate distinct dysfunctional HDL forms of potential clinical prognostic utility.